Ductal carcinoma in situ (DCIS) is an early-stage form of breast cancer that accounts for over 20% of all breast cancers in the US. This proposal aims to elucidate the underlying molecular and pathological mechanisms of DCIS. Our recent work revealed that the expression of miR29c in tumor-initiating cells was significantly down- regulated in DCIS compared to normal cells. Furthermore, miR29c suppressed expression of the ID2 gene, a dedifferentiation factor, in both non-tumorigenic and premalignant cells. We also found that ID2 contributed to formation of tumor-initiating cells in DCIS. As a transcription regulator, ID2 controls gene expression by sequestering bHLH transcription factors; we found that ID2 up-regulated expression of both the SOX2 and fatty acid synthase (FASN) genes. SOX2 is a well- known self-renewal regulator of stem cells, while FASN is a key enzyme for lipid metabolism and plays a critical role in tumor progression. We also found that the FASN gene is significantly up-regulated in tumor-initiating cells of DCIS and that resveratrol, a compound found abundantly in grapes, suppressed FASN expression and blocked growth of DCIS. Among resveratrol derivatives, the small-molecule drug pterostilbene was ten times more effective than others in suppressing FASN. Based on these novel discoveries, we hypothesize that (a) DCIS is initiated from tumor-initiating cells in a process controlled by the miR29c-ID2 axis, (b) ID2 promotes self-renewal of tumor-initiating cells by regulating SOX2 and FASN through sequestration of bHLH transcription factors; and (c) pterostilbene prevents DCIS formation by suppressing FASN expression. To test these hypotheses, we propose a series of experiments to: determine the mechanisms of the molecular pathway and validate in clinical samples (Aim 1); clarify the role of miR29c and ID2 in DCIS initiation in various animal models (Aim 2); and then test the potential therapeutic utility of selected small molecule compounds in DCIS prevention (Aim 3). The long-term objective of this proposed research is to develop a novel therapeutic strategy for DCIS prevention, which will have a significant impact on current clinical practice. Training will be provided to the applicant through relevant course work, weekly seminars and journal club presentations, one-to-one meetings with the primary mentor, and critical analysis and discussions of the applicant's progress with the mentor team. In addition, the research training program is designed to enable (i) generation of conceptual frameworks, (ii) exploration of cutting-edge technology to execute the planned project, (iii) enhancement of grant- and manuscript-writing skills, and (iv) building a network with top-notch cancer researchers through scientific meetings. Both the state-of-art facilities available at the Comprehensive Cancer Center of Wake Forest University and the seasoned mentor will facilitate the applicant's progress toward becoming an independent investigator who conducts clinically meaningful translational research in cancer biology.